Several different phospholipid transfer proteins have been identified (Phospholipid transfer proteins from lung, properties and possible physiological functions, Chemistry and Physics of Lipids, Volume 38, Issues 1-2, 30 Aug. 1985, Pages 17-27 Jane D. Funkhouser and Robert J. Readand) these are expressed in different tissues in addition to the more familiar plasma PLTP, which is a 51 kD protein containing 476 amino acid residues and is found mostly in plasma, placenta and pancreas, but is also present in lung, kidney, heart, liver, skeletal muscle, small intestine and brain. For example, two different PLTP's have been found in lung and they differ significantly from the plasma PLTP. (Properties of a non-specific phospholipid-transfer protein purified from rat lung, Biochimica et Biophysica Acta (BBA)—Lipids and Lipid Metabolism, Volume 752, Issue 1, 16 Jun. 1983, Pages 118-126)
The nucleotide sequence encoding of the plasma PLTP has been cloned and sequenced as disclosed in U.S. Pat. No. 5,610,019, the contents of which are incorporated herein by reference. The activity of this protein has been reviewed by Tall, in An. Rev. Biochem. (1995) 64:235-237.
Phospholipid transfer proteins facilitate transfer of substrates which include phospholipids, diglycerides and vitamin E from donor unilamellar vessels or lipoproteins into HDL or other lipoproteins. It is also known that the plasma PLTP plays a role in HDL formation and in regulating the secretion of Apo-B containing lipoproteins. Plasma PLTP also plays a role in atherosclerosis development and Cholesteryl ester transfer protein (CETP) activity is enhanced by the plasma PLTP.
With respect to assays for PLTP activity, the above-referenced '019 patent describes a heterogeneous assay where the acceptor is biotinylated HDL. In a study involving assessing the substrate specificity of PLTP, in particular in investigating whether PLTP would interact with LPS, Hailman, E., et al., J: Bioi. Chem. (1996) 271:12172-12178 describe an assay in which LPS labeled with the fluorophore boron dipyrromethane difluoride (BODIPY) is transferred from self-associated micelles to various acceptors. The authors found that PLTP could mediate the exchange to HDL particles but not to CD14.
Oskolkova, O. V., et al., Chem. Phys. Lipids (1999) 99:73-86 employed pyrene conjugates of phospholipid-coupled thymidine to monitor, the spontaneous transfer of thymidine from vesicles to acceptors; high levels of spontaneous transfer were observed.
Pyrene was also used as a label by Huuskonen, J., et al, Biochem/Biophys. Acta (1996) 1303:207-214. These authors studied the specificity of the enzyme using pyrene-labeled phospholipids from quenched donor phospholipid vessels to HDL3 particles.
Lalanne, F., et al., J. Lipid Res. (2001) 42:142-149, in studying the modulation of transfer of phospholipids by diacylglycerols also employed pyrene-labeled phosphatidylcholine and measured transfer from various types of vesicles to HDL.
While pyrene is often referred to as self-quenching, this is not in fact the case. Pyrene has a monomer/excimer emission profile whereby at low concentrations the monomer fluoresces at 390 nm but at higher concentrations the emission shifts to 470 nm. The ratio of 390/470 intensities is measured; however, this ratio is also dependent on viscosity and temperature. Thus, pyrene is not truly a self-quenching fluorophore.
Nichols, J. W., Seminars in Cell and Developmental Biology (2002) 13: 190-184 measured trafficking of phospholipids using N-nitrobenz-2-oxa-1,3-diazol 4-yl (NBD) as label. The paper does not concern an assay for levels of PLTP; rather, the movement of phospholipids in various contexts is measured.
Finally, U.S. patent application Ser. No. 10/279,787, filed Oct. 23, 2002 by this inventor discloses a PLTP activity assay which is improved upon in the current invention.
The measurement of PLTP activity has been handicapped by an extremely high background of spontaneous transfer from donor to acceptor. In addition, many prior art assays are heterogeneous assays and thus require separation of donor from acceptor before measurement is made.
The present invention minimizes the background of spontaneous transfer; in one embodiment, the assay is conducted as a homogeneous assay, thus offering added convenience. The invention further addresses the issue of substrate specificity by providing the ability to measure and express specific transfer rates of various substrates. The invention proves useful in the characterization of diseased states.